Antigenic peptides

ABSTRACT

A method for designing antigenic peptide libraries accounts for naturally occurring and potential variability in a group of protein sequences from a variable pathogen. The peptide libraries can elicit an immune response against a range of pathogen variants.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government may have certain rights in this invention pursuantto Department of Health and Human Services Grant Nos. ISTC #2450 andBTEP #34.

CLAIM OF PRIORITY

This application claims priority to Russian Patent Application SerialNo. 2002126396, filed on Sep. 27, 2002, the entire contents of which arehereby incorporated by reference.

TECHNICAL FIELD

This invention relates to antigenic peptides.

BACKGROUND

A number of pathogens important to human health are variable pathogens,such as HIV-1, HIV-2, hepatitis B and C, influenza, dengue types 1-4viruses, malaria, and tuberculosis. These and other variable pathogenspresent a challenge to vaccine development, due to the extremely highgenetic variability of the pathogen. In particular, the number of peopleinfected with HIV throughout the world is growing rapidly, especially incountries with poor health care resources. The development of a highlyefficient vaccine could help restrict the propagation of this epidemic.One of the main obstacles to developing such a vaccine is the extremelyhigh genetic diversity of HIV. The genetic diversity is observed invarious regions of the world in different subtypes, within a singlesubtype and even, in some cases, within an individual. This variabilityresults from the high rate of mutations in viral proteins occurringduring virus replication. See, for example, Coffin, J. M. Science 1995,267:483-489; Delwart et al., J. Virol. 1994 68:6672-6683; Markham R. B.et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95:12568-12573; andMcCutchan, F. E. et al. AIDS 1996 10 Suppl 3:S13-S20, each of which isincorporated by reference in its entirety.

It has been shown in animal models that simultaneous exposure tomultiple antigenic determinants often induces immune responses to eachof them (Thomson, S. A. et al. Proc. Natl. Acad. Sci. U.S.A. 1995,92:5845-5849; Thomson, S. A. et al. J. Virol. 1998, 72: 2246-2252; andWoodberry, T. et al. J. Virol. 1999, 73:5320-5325, each of which isincorporated by reference in its entirety). This indicates that multipleantigenic determinants are suitable as components of a single vaccineconstruct.

SUMMARY

The protective component of an immune response to a variable pathogen isoften directed against a variable region of a protein. If, on the otherhand, the protective response was directed against a conserved region ofa protein, pathogen variability would not present a problem toimmunological control of infections and vaccine prophylaxis. However, animmune response against conserved regions of variable pathogens oftenproves to be nonprotective. A peptide antigen based on a variable regioncan induce a protective response, but the variability of these regionsis so great that it is impractical to cover the variability by additionof individual peptide sequences. A potential vaccine against a variablepathogen should include a variety of antigens, to produce an immuneresponse to the variants of the pathogen to which an individual maybecome exposed.

Vaccine prophylaxis or therapy against variable infectious agentsrequires constructing a vaccine capable of inducing wide-range humoraland cellular immune responses to antigen variable regions. A challengefor vaccine prophylaxis and therapy against variable infectious agentsis the development of a vaccine capable of inducing an immune responseagainst variable or hypervariable regions of their protein components.

A library of chimeric peptide antigens containing one or morepotentially protective epitopes and vaccine constructs containing suchantigens can produce a protective immune response. In particular,chimeric peptide libraries are able to induce a protective immuneresponse including both humoral and cellular responses to HIV-1, HIV-2,hepatitis C and B viruses, influenza virus, dengue types 1-4 viruses,malaria, tuberculosis, or other variable pathogens.

Chimeric peptide libraries can be designed to represent naturallyoccurring or potential variants of protein epitopes. Peptide librariesdeveloped in this manner can produce a broad immune cross-response andcan be applied for constructing vaccines, pharmaceuticals, anddiagnostic kits. The design methods can be applied to a broad range ofantigenic determinants of an infectious agent. Different methods can beapplied to one infectious agent or one of its proteins or to a singleepitope of the protein.

Immunogenic sets of chimeric peptides, referred to as variable chimericpeptide libraries (VCPLs), represent the naturally occurring andpotential variability of antigenically active regions. Variable chimericpeptide libraries are sets of homologous peptides variable at one ormore positions. They are designed to mimic the genetic diversity ofvariable protective determinants of an infectious agent. Such VCPLs caninduce production of a wide range of antibodies and cytotoxicT-lymphocytes (CTLs) with a joint specificity that covers the diversityof antigenic variants of the variable infectious agent.

In one aspect, a method of manufacturing a family of antigenic peptidesincludes locating a plurality of variable positions in a region of apathogen protein, choosing a peptide sequence of the pathogen proteinincluding the plurality of variable positions, selecting one or moresubstitute amino acid residues for one of the variable positions basedon antigenic similarity to amino acid residues naturally occurring atthe variable position of the pathogen protein, and preparing a family ofantigenic peptides based on the peptide sequence and including thesubstitute amino acid residues.

In another aspect, a method of designing a family of peptide sequencesincludes locating a plurality of variable positions in a region of apathogen protein, choosing a peptide sequence of the pathogen proteinincluding the plurality of variable positions, and selecting one or moresubstitute amino acid residues for one of the variable positions of thepeptide based on antigenic similarity to amino acid residues naturallyoccurring at the variable position of the pathogen protein, therebyforming a family of peptide sequences.

Selecting can include determining the antigenic similarity using anantigenic similarity matrix. A frequency can be assigned to eachsubstitute amino acid residue in the family of antigenic peptides.Preparing can include weighting the substitute amino acid residues inthe family of antigenic peptides based on the assigned frequency.Assigning can include considering the frequency with which thevariations naturally occur. The pathogen protein can include ahypervariable region. The pathogen protein can be associated with avirus. The virus can be HIV, hepatitis B virus or hepatitis C virus, oran influenza. The pathogen protein can be HIV gp120. The region can bethe V1, V2, V3, V4, or V5 regions. The pathogen protein can beassociated with a malaria pathogen, a dengue pathogen, or a tuberculosispathogen.

The family of antigenic peptides can include members, such that themembers taken together have antigenic similarity to each naturallyoccurring sequence of the region of the pathogen protein. The family ofantigenic peptides can include members, such that the members takentogether have antigenic similarity to a non-naturally occurring sequenceof the region of the pathogen protein. The method can includeidentifying peptide sequences of the family, the identified peptidesequences being representative of the sequence diversity of the entirefamily. Fewer than 500 sequences can be identified as beingrepresentative of the sequence diversity of the entire family.Identifying can include calculating a distance between peptide sequencesof the family. Calculating a distance can include using an antigenicsimilarity matrix.

The method can include determining an antigenic similarity between apeptide of the family and a region of a human protein. The method caninclude removing a peptide from the family of antigenic peptides beforepreparing the family if the determined antigenic similarity between thepeptide of the family and the region of a human protein exceeds apredetermined threshold.

Preparing the family of antigenic peptides can include chemicalsynthesis of the family of peptides. The chemical synthesis can includescombinatorial synthesis, whereby the peptides are formed as a mixture ofdifferent sequences. The separate peptides can be mixed. The chemicalsynthesis can include parallel synthesis, whereby each peptide is formedseparately from other peptides. Preparing the family of antigenicpeptides can include expression of the family of peptides by a hostorganism.

In another aspect, a composition includes a family of antigenic peptideshaving amino acid sequences having antigenic similarity to amino acidsequences of a variable region of a pathogen protein, wherein eachantigenic peptide in the family has at least one amino acid positionthat varies relative to other antigenic peptides in the family.

One amino acid residue can occur more frequently than another in theposition that varies. The family can include greater than 150 or greaterthan 1,000 mutually unique antigenic peptides. The family can includefewer than 100,000 or fewer than 50,000 mutually unique antigenicpeptides. The family can include between 1,000 and 50,000 mutuallyunique antigenic peptides. The family of antigenic peptides can includesequences having antigenic similarity to sequences from a subtype ofHIV. The subtype can be subtype A, subtype B, subtype C, subtype D,subtype F, subtype G, a recombinant subtype, a subtype of HIV group N, asubtype of HIV group O, or combinations thereof. At least two members ofthe family of antigenic peptides can be mixed together. The family ofantigenic peptides can be separated according to sequence. The familycan include a multiple antigenic peptide.

In another aspect, a method of eliciting an immune response in a subjectincludes administering to the subject a composition including a familyof antigenic peptides having amino acid sequences having antigenicsimilarity to amino acid sequences of a variable region of a pathogenprotein of a pathogen.

Antigenic similarity can be determined using an antigenic similaritymatrix. The composition can be administered to a subject prior toinfection by the pathogen. The family of antigenic peptides can haveamino acid sequences having antigenic similarity to amino acid sequencesof a subtype of the pathogen protein. The family of antigenic peptidescan have amino acid sequences having antigenic similarity to amino acidsequences from more than one subtype of the pathogen protein. Thecomposition is administered to a subject infected by the pathogen. Thesubject can be infected by a subtype of the pathogen. The family ofantigenic peptides can have amino acid sequences having antigenicsimilarity to amino acid sequences from the subtype by which the subjectis infected.

In another aspect, a method of diagnosing infection includes contactinga sample with a family of peptides having amino acid sequences havingantigenic similarity to amino acid sequences of a variable region of apathogen protein, wherein each peptide in the family has at least oneamino acid position that varies relative to other peptides in thefamily.

The family of peptides can be antigenically similar to a pathogensubtype, or to more than one pathogen subtype. The family of peptidescan be immobilized on a substrate before contacting. The method caninclude determining if the sample includes antibodies that bindspecifically to the family of peptides.

In general, antigenic peptide libraries reflect the existing andpotential diversity of a variable region of a pathogen protein. Thepeptide libraries can be used in prophylactic, therapeutic, anddiagnostic applications. The full antigenic diversity of a pathogenepitope can be represented by the library. A library designed to reflectthe diversity of all variants of a pathogen can be a broad-spectrumvaccine or diagnostic. A library designed to reflect the diversity oneor more subtypes of pathogen can be a subtype-specific vaccine ordiagnostic. Peptide sequences that are highly similar to sequences foundin human proteins and are thus likely to induce an autoimmune reactioncan be removed from the library. A library can be designed for anyvariable pathogen protein of known sequence. Examples of variablepathogens include HIV-1, HIV-2, hepatitis B and C, influenza, denguetypes 1-4 viruses, malaria, and tuberculosis.

The details of one or more embodiments are set forth in the descriptionbelow. Other features and advantages will be apparent from thedescription and from the claims.

DETAILED DESCRIPTION

Much effort has been devoted to development of a vaccine against HIV inparticular, but without success to date. To be efficient against thebroad range of HIV-1 isolates, potential vaccines should containimmunogens of main isolates representing the entire range of HIV-1isolates. It is necessary to identify these immunogens and improve theirimmunogenic properties (McMichael, A. J., and Hanke, T, Nature Medicine2003, 9:874-880; Baltimore, D. and Heilman, C. Sci. Am. 1998, 279:98-103; and van der Groen, G. et al. AIDS Res. Hum. Retroviruses 1998,14 Suppl. 3: S211-S221, each of which is incorporated by reference inits entirety).

Therefore, the resulting vaccine construct should contain key protectiveB- and T-cell epitopes specific to the broad range of HIV-1 isolates,combined with modern delivery means and adjuvants. Activation of bothhumoral and cellular branches of the immune system is required(Berzofsky, J. A., and Berkower, I. J., AIDS 1995, 9 Suppl. A:S143-S157, which is incorporated by reference in its entirety). Thelocal immune response on mucous membranes and circulating neutralizingantibodies are considered to play key roles in preventing HIV infection,whereas virus-specific CTLs are likely to eliminate infected cells. See,for example, Lehner, T. et al. Science 1992, 258:1365-1369; and Goulder,P. J. and Walker, B. D. Nature Med 1999, 5:1233-1235, each of which isincorporated by reference in its entirety.

The amino acid sequence of the envelope glycoprotein (also called env orgp120) of HIV is highly variable between independent isolates andbetween sequential isolates from a single infected individual. The aminoacid variability in gp120 is concentrated in specific variable regions,numbered V1-V5. The most variable regions often contain neutralizingepitopes so that the virus partially evades the host's immune responseand establishes a persistent infection. The principal neutralizingdeterminant of gp120 is located in the third hypervariable region (V3).

A number of candidate peptide vaccines against HIV-1 have been developedwith the recognition of the V3 region as the principal neutralizingdeterminant in mind. For example, a method of rational design of apolyvalent subunit vaccine against HIV included determination ofneutralizing epitopes of the V2 and V3 domains of HIV gp120 isolatesfrom various areas of the world. See, for example, U.S. Pat. No.6,042,836, which is incorporated by reference in its entirety. Anotherapproach to synthetic immunogenic HIV-1 peptides included designing atandem synthetic peptide containing the sequences of T- and B-cellepitopes and the sequence of the V3 loop from various HIV isolates. See,for example, U.S. Pat. No. 5,817,754, which is incorporated by referencein its entirety. However, the high genetic variability of the V3 domainresults in a variety of sequences, which cannot be simultaneouslyintroduced into a vaccine construct.

For the same reasons as in the case of vaccines, it is extremelydifficult to diagnose antibodies against hypervariable regions ofvariable infectious agents. These antibodies are the earliest anddominant in the immune response. An example is antibodies against the V3domain of HIV-1 gp120. See, for example, Baltimore, D. and Heilman, C.Sci. Am. 1998, 279: 98-103, Berzofsky, J. A., and Berkower, I. J., AIDS1995, 9 Suppl. A: S143-S157, and Carlos, M. P. et al., AIDS Res. Hum.Retroviruses 2000, 16:153-161, each of which is incorporated byreference in its entirety.

The diagnostics of such antibodies against the V3 hypervariable domainof gp120 depends on the genotypes of the antigens involved and thegenotype of the infectious agent. It can require using multiplesequences corresponding to a broad range of HIV-1 variants. Otherwise,there is a high risk of missing a rare or modified variant of the virus.

A peptide-based vaccine can include a library of peptides. The peptidescan be similar to a variable region of a pathogen protein. Inparticular, the variable region can include a known antigenicdeterminant. The peptide sequences in the library are chosen to reflectthe naturally occurring and potential variability of the variableregion.

Chimeric peptide libraries that mimic the genetic diversity of variableprotein regions of a pathogen can induce a protective immune response toa broad spectrum of pathogen variants. To design the chimeric peptidelibrary, a theoretical analysis of protein sequence information iscarried out. The theoretical analysis results in a library design thatcan mimic all known and potential variants of the variable proteinregion. The library can be a subtype-specific library, mimicking onlythose variants belonging to a particular subtype of the pathogen, or abroad spectrum library, mimicking variants belonging to more than onesubtype. The peptides of the library can bind antibodies that arespecific to its various antigenic variants.

In general, the design of an antigenic chimeric peptide library beginsby inspecting sequences of a protein from a variable pathogen. Avariable region of the protein is identified. The variable region can bea known antigenic determinant. A peptide fragment within the proteinsequence including at least one variable position is selected to be thebasis of the chimeric peptide library. The fragment includes one or morepositions having naturally sequence variations. The peptide fragment caninclude non-varying positions. The fragment can be 10-50 residues inlength, such as 15-45 residues or 20-40 residues in length. For eachvariable position in the peptide, the residues that occur in thatposition and the frequency with which each occurs are identified. Thechoice of residues at each variable position in the library isdetermined by considerations of antigenic similarity to the residuesnaturally occurring at that position. The frequency of each residue at avariable position is assigned according to the frequency that it or itscorresponding antigenically similar residue occurs naturally. Selectionof residues and frequencies is described in more detail below. Theresidue selection and frequency assignment can be automated, i.e.carried out by a computer program.

The number of sequences in the library (the size of the library) is theproduct of the number of possible residues at each position in thepeptide. The size of the library can include, for example, more than200,000,000 sequences, fewer than 200,000,000 sequences, fewer than500,000 sequences, fewer than 200,000 sequences, fewer than I 00,000sequences, fewer than 50,000 sequences, more than 5,000 sequences, morethan 10,000 sequences, or more than 20,000 sequences. The size of thelibrary can be reduced by eliminating the residue that occurs with thelowest frequency until a desired size is reached.

Immunogenic sets of chimeric peptides, (variable chimeric peptidelibraries or VCPLs) when taken together, represent the naturallyoccurring and potential variability of antigenically active regions.Variable chimeric peptide libraries are sets of homologous peptidesvariable at one or more positions. They are designed to mimic thegenetic diversity of main variable protective determinants of aninfectious agent. Such VCPLs can induce a wide range of antibodies andcytotoxic T-lymphocytes (CTLs), having a joint specificity that coversthe diversity of antigenic variants of the infectious agent. Thus, VCPLsinduce humoral and cellular immune responses against the diversity ofcurrently existing variants of an infectious agent and against potentialvariants which may arise in the future.

Libraries of variable chimeric peptides can be constructed so that theconserved positions of these peptides represent amino acids common forall sequences, and the heterogeneous diversity of the variable positionsis mimicked by one or several amino acids. The mimicking amino acids canbe chimeric, in other words, they mimic the general heterogeneity ofvariable positions but need not occur at corresponding positions amongthe known and potential diversity of natural sequences.

The method of designing VCPLs rests upon comprehensive investigation ofthe whole body of structural evidence on the organization ofimmunodominant protein epitopes and on the induction of a protectiveimmune response to the target pathogen. The library design begins withcollection and analysis of amino acid sequences from the targetpathogen, and estimation of the genetic variability and epidemiologicalsignificance of pathogen subtypes. The amino acid sequences are searchedfor conserved, variable, and hypervariable regions that contribute tothe development of a protective immune response against the pathogen.

The antigenic properties of the peptide library can be controlled byselecting the pathogen protein sequences that the library is designedfrom. For example, a library mimicking a particular subtype of pathogencan be designed by selecting only sequences derived from that subtype.In general, the sequences within any one subtype or sub-subtype are moresimilar to each other than to sequences from other subtypes throughouttheir genomes. Group M is the main group of viruses in the HIV-1 globalpandemic, and it contains multiple subtypes. Group O is the “outlier”group, and group N is a very distinctive form of the virus that isNon-M, Non-O. The HIV-1 subtypes from the M group of HIV-1, for example,are phylogenetically associated groups or clades of HIV-1 sequences, andare labeled A1, A2, B, C, D, F1, F2, G, H, J and K. In addition,recombinant HIV-1 sequences are possible, where the viral genomeincludes regions of sequence from more than one subtype. See, forexample, the HIV Sequence Database, at hiv-web.lanl.gov. These subtypesrepresent different lineages of HIV, and have some geographicalassociations. A library mimicking a particular subtype or recombinantform can be useful, for example, in cases where particular subtypes arefound predominantly in particular geographic locations.

The number of potential evolutionary variants of the sequence of anantigenically active region can be enormous. For example, the potentialantigenic variability of the V3 region of HIV-1 gp120 is as high as10²⁵-10³⁰ variants. Once the complete variability at each position inthe peptide is determined from the naturally occurring sequences, thevariability of the desired VCPL can be reduced by recognizing the mostsignificant amino acids that determine the genotypic diversity of theantigenic variants. The rates of occurrence of various amino acids atvariable positions of an immunogenic epitope are determined with regardto their relative occurrence in the alignment of the sequences of theregion at issue.

The size of the VCPL can be decreased by recognizing the mostantigenically significant amino acids at each variable position, andcombining them in groups of variable composition with one or more aminoacids representing the groups of variable composition. The amino acidsrepresenting groups of variable composition are determined on the basisof antigenic similarity.

Antigenic similarity is a quantitative measure of how readily one aminoacid residue can substitute for another in a protein-proteininteraction, such as, for example, an antibody-antigen interaction. Tworesidues are very antigenically similar if substituting one for theother in a particular epitope has a small effect on the antigenicbehavior of that epitope. Conversely, if the substitution has a largeeffect, then the two residues have a low antigenic similarity. Ingeneral, residues that have similar physical and chemicalcharacteristics will have a high antigenic similarity.

Antigenic similarity can be expressed in a matrix, called an antigenicsimilarity matrix (ASM). The construction of a matrix is described in,for example, Maksyutov, A. Z., Eroshkin, A. M., and Kulichkov, V. A,Mol. Biol. 1987, 21:30-47, which is incorporated by reference in itsentirety. The matrix can account for the affinity of two residues, thatis, how frequently two residues are in contact in a globular protein, asdetermined by examination of 3D structures. See, for example, Warme, P.K., and Morgan, R. S., J. Mol. Biol. 1978, 118: 289-304, which isincorporated by reference in its entirety. The matrix is also adjustedfor the frequency with which an amino acid occurs in the hypervariableregions of immunoglobulins. See, for example, Bourgarit, J., J. Ann.Immunol. 1980, 131D: 267-287, which is incorporated by reference in itsentirety.

The values of an initial matrix can be transformed by the formula:ASM _(ij)=100−(R _(ij) +R _(ji))/2for each pair of amino acids i and j, and R_(ij) and R_(ji) are elementsof the initial matrix, thus producing a symmetric matrix. Todifferentiate the effects of coinciding amino acids and increase theweight of the residues that occur frequently in antigenic epitopes, thediagonal elements can be adjusted according to the following formula:ASM _(ii) =ASM _(ii)+5(h _(i) +s _(i) +c)where h_(i) is the hydrophilicity of the amino acid (Hopp, T. P., andWoods, K. R., Proc. Natl. Acad Sci. USA 1981, 78:3824-3828, which isincorporated by reference in its entirety), s_(i) is the relativefrequency of the amino acid in antigenic determinants of proteins(Maksyutov, A. Z. and Zagrebelnaya, E. S., Comp. Applic. Biosci. 1993,9:291-297, which is incorporated by reference in its entirety), and c is6.6, the minimum value of h_(i)+s_(i), introduced to insure a positivesum (h_(i)+s_(i)+c). Finally, to satisfy the local alignment programSIM, the quantity 75 is subtracted from each element in the matrix. Onesuch matrix is presented in Table 1. The ASM of Table 1 is aimed at thesearch for locally similar regions exhibiting close structural featuresin respect to interaction between an antibody or another binding ligandand the binding site (see, for example, Maksyutov A. Z. et al.,Molecular Biology 2002, 36: 346-358, which is incorporated by referencein its entirety).

Qualitatively, a matrix value for a pair of amino acids indicates thatthey are good substitutes for one another, and a low number indicatesthat they are poor substitutes. For example, valine and leucine have ahigh antigenic similarity according to the matrix (matrix value of 9),but valine and aspartic acid do not (matrix value of −59), as would beexpected from chemical considerations. TABLE 1 A C D E F G H I K L M N PQ R S T V W Y A 59 C −15 50 D −41 −50 81 E −49 −53 4 79 F −7 −12 −49 −5147 G 5 −15 −25 −35 −25 49 H −30 −34 −42 −49 −34 −35 61 I 0 −22 −62 −65−4 −35 −48 37 K −38 −49 −48 −62 −46 −35 −12 −55 88 L 3 −12 −55 −60 4 −30−41 11 −47 48 M −7 −15 −57 −55 −5 −30 −33 −3 −46 4 37 N −36 −37 −20 −27−44 −15 −23 −57 −23 −46 −43 55 P −11 −13 −28 −36 −17 −15 −8 −29 −24 −19−19 −19 59 Q −24 −24 −15 −29 −29 −10 −11 −43 −31 −34 −30 3 −10 71 R −34−42 −44 −54 −39 −25 −13 −51 7 −46 −45 −23 −24 −26 69 S −23 −31 −15 −35−34 −5 −3 −45 −19 −38 −40 −10 −4 −5 −15 61 T −27 −30 −20 −26 −33 −10 −18−46 −31 −41 −42 −15 −13 −11 −18 6 62 V −1 −16 −59 −62 −4 −30 −44 13 −559 −1 −51 −26 −37 −50 −42 −39 47 W −22 −21 −38 −39 −18 −40 −31 −27 −44−19 −22 −23 −9 −19 −38 −23 −17 −19 24 Y −25 −30 −26 −20 −20 −25 −25 −37−31 −27 −23 −10 −13 −12 −32 −19 −18 −32 −8 53

At each position in the peptide, the matrix value for each pair ofresidues occurring naturally is looked up. A pair having a positivematrix value will be represented in the library by the residue thatoccurs more frequently in the natural sequences, and its frequency inthe library will be the sum of frequencies for each residue that itrepresents. The remaining residues are combined in subgroups, and foreach subgroup, the matrix is searched for a residue that has a positivematrix value with each residue in the subgroup. If such a residue isfound, it is assigned a frequency equal to the sum of frequencies ofresidues in the subgroup. This residue can be one that does notnaturally occur in that position. If a naturally occurring residue doesnot have a positive matrix value with any other naturally occurringresidue at that position, and cannot be represented by another residuenot naturally occurring at that position, it will represent itself inthe library.

As an example of how the matrix can be used to choose residues for aparticular 15 position in a peptide library, consider a variableposition in where the following six different residues occur naturally:A (5%), D (20%), E (35%), M (22%), V (10%), and Y (8%). The matrix valuefor each pair of naturally occurring residues is then looked up: AD −41AE −49 AM −7 AV −1 AY −25 DE 4 DM −7 DV −59 DY −26 EM −55 EV −62 EY −20MV −1 MY −10 VY −32

Only one pair, DE, has a positive matrix value. That pair of residueswill be represented by the more frequent naturally occurring residue, E,and will have a frequency equal to the sum of the frequency of theresidues it represents (20+35=55%). The remaining residues, A, M, V andY, are divided into subgroups and the matrix is searched for otherresidues that are antigenically similar to these. The subgroup ofresidues A, M, and V each have a positive matrix value with L: AL 3 ML 4VL 9

Thus, in the library, A, M, and V will be represented by L, which isantigenically similar to all three. The frequency of L in the library isthe sum of the frequencies of A, M and V: 5+10+22=37%. Finally, since Yis not antigenically similar to another residue, Y represents itself inthe library with a frequency of 8%. Table 2 summarizes how the residuesfrom natural sequences are represented in the library for this example.TABLE 2 Naturally occurring Library D 20% E 55% E 35% A  5% L 37% M 22%V 10% Y  8% Y  8%

Theoretical analysis of the potential cross-reactivity of antibodiesraised against peptide libraries shows that in order to induce an immunecross-response sufficiently broad to cover the whole set of antigenicvariants of a variable infectious agent would require a librarycontaining tens of thousands of homologous peptides. A smaller libraryhaving hundreds of homologous peptides could not induce a sufficientlybroad immune response to existing and potential variants of a variableinfectious agent. However, increasing the size of the library alsoincreases the danger of that the library will elicit an autoimmuneresponse. The analysis was carried out by methods for estimation of thecross-reactivity of antibodies against homologous antigens. See, forexample, Maksyutov A. Z., Voprosy Virusologii 1989, 3: 283-288;Maksyutov A. Z., Eroshkin A. M., and Kulichkov V. A., MolekularnayaBiologiya 1987, 21: 3947; Maksyutov, A. Z., and Zagrebelnaya, E. S.,Computer Applications in the Biosciences 1993, 9: 291-297; andMaksyutov, A. Z., and Zagrebelnyi S. N., Molekularnaya Biologiya 1993,27: 980-991, each of which is incorporated by reference in its entirety.

Typically during the natural infection of an individual and selection ofantigenic variants of an infectious agent there is too little time forformation of a sufficient amount of immune effectors capable ofcross-reaction with normal human proteins to induce an autoimmuneresponse. Induction of autoimmune processes usually takes a long time,and often requires the constant presence of the sensitizing antigen.Hence, the probability of autosensitization in an individual as a resultof local similarity between variants of the hypervariable regions of thevariable infectious agent and a human protein may be insignificant. Onthe other hand, the risk of induction of autoimmune conditions isincreased by repeated use of a peptide vaccine for prophylaxis ortherapy of an infectious disease. Therefore, during the design ofpolyepitope vaccine constructs, it should be taken into account that thelong persistence of “time-conservative” variants of variable regions ofthe variable infectious agent can bring about autoimmune conditions,particularly with a vaccine construct containing adjuvants forincreasing the immune response. Thus, the design of vaccine constructson the basis of VCPLs requires comprehensive theoretical considerationof the local similarity between VCPL peptides and the whole set of humanproteins.

It can be advantageous to screen the sequences in a library for theirpotential to generate an autoimmune response. If a peptide in thelibrary is highly similar to a peptide present in a human protein, animmune response could be generated against the human protein, withdeleterious health effects for the person. A measure of the likelihoodthat a peptide sequence might generate an autoimmune response can becalculated by determining the antigenic similarity of the peptidesequence in the library to sequences found in human proteins.

At the final stage of the method of designing a VCPL, the localsimilarity between the VCPL peptides and the whole set of human proteinscan be analyzed. Candidate vaccines on the based on VCPLs can bescreened for potentially autoimmunogenic determinants resulting fromlocal similarity of VCPL peptides to human proteins. These potentiallyautoimmunogenic determinants can be removed from the library.

The epitopes to be modeled in a VCPL can be chosen so as not to beautoimmunogenic. The method of identifying potentially immunopathogenicregions in proteins of an infectious agent on the basis of recognitionof regions displaying close local similarity to human proteins providesa key to rational design of safe polyepitope vaccines against pathogenicmicroorganisms, because it allows elimination of epitopes with animmunopathogenic potential. In particular, antigenic determinantsdisplaying similarity to human proteins that occur often and/or supportimportant physiological functions of the body should be eliminated.

To search for local similarity between a library sequence and humanproteins, the SIM program can be used (Huang, X. and Miller, W., Adv.Appl. Math., 1991, 12, 337-357, which is incorporated by reference inits entirety). The sequences of human proteins are available in publicdatabases, for instance SWISS-PROT (rl. 41, FEB-2003) and SP-TrEMBL (rl.23, MAR-2003). The comparison can be carried out using the version ofSIM available on the European Molecular Biology Laboratory (EMBL)server. The following parameters of the SIM program can be used:gap-open penalty 100, gap-extension penalty 20.

The local similarity search may produce a large number of hits, many ofwhich have a small degree of similarity between a peptide sequence fromthe library and a region of a human protein. It can be useful to filterthe results of the similarity search to identify most relevantpeptide-protein similarities. The filtering can be performed as follows.The twenty commonly occurring amino acids are divided into intersectinggroups with physicochemical similarities: group 1-Lys, Arg; group 2-Asp,Glu; group 3-Asn, Gln; group 4-Ser, Thr; group 5-Ala, Ile, Leu, Val;group 6-Ala, Gly; group 7-Leu, Phe; group 8-Leu, Met, Val; and group9-Phe, Trp, Tyr.

For each individual hit of local similarity between a library peptideand a human protein, the following values are calculated: index ofsimilarity (Score determined by the SIM program); the percentage ofmatching amino acids (Match %); the number of matching amino acids (M);the number of mismatching amino acids (MM); the number of gaps (Gap);the number of conservative substitutions (C_(cons)) (that is, the numberof substitutions within groups 1, 2, 3 & 4, 5-9); the number ofsubstitutions of charged amino acids (His, Lys, Arg, Asp, Glu) by one ofopposite charge (C⁺⁻); the number of non-conservative substitutions ordeletions of charged amino acids (C_(±)); the number of non-conservativesubstitutions or deletions of polar amino acids (Asn, Gln, Ser, Thr)(C_(polar)); and the number of non-conservative substitutions ordeletions of aromatic amino acids (Phe, Trp, Tyr) (C_(arom)).

Then an empirical algorithm tests if the following conditions are met:

-   -   1) Index of similarity not less than 320 (Score≧320).    -   2) Number of matching amino acids in a given region not less        than 6 (M≧6).    -   3) Percent of matches not less than 50% (Match % ≧50%).    -   4) Number of matching amino acids and conservative substitutions        in a given region not less than 8: M+C_(cons)≧8. However, if        within a homologous region there is a subregion which comprises        a string of 8 amino acids in a row, which are either        conservative or matching then this region is taken as a whole        regardless of the percent of match (Match %).    -   5) (MM−C_(cons)+Gap)/(M+C_(cons))≦{0.37|_(m=6+∞)}    -   6) 1.5*C⁺⁻+C_(±)+0.5*(C_(polar)+C_(arom))≦0.1+0.5*(M+C_(cons)−6)

If all the above criteria are met, then the nature and/or function ofthe human protein can be considered. If the library sequence is similarto a sequence that occurs frequently in human proteins, or is similar toa sequence in a protein having an important physiological function, thatsequence can be removed from the library.

In certain circumstances, it can be desirable for the library to have asmall number of sequences, such as fewer than 1,000 sequences, fewerthan 500 sequences, or fewer than 250 sequences. The library isrepresentative of the full antigenic diversity found in the naturallyoccurring sequences. Such a representative chimeric peptide library(RCPL) can be created by selecting sequences from a large library (e.g.,a VCPL) such that the selected sequences are representative of the fullantigenic diversity found in the naturally occurring sequences andnon-natural sequences generated from the large library. The non-naturalsequences can reflect potential sequences, for example, by including ina single sequence variants at different positions that have not beenfound together naturally.

Because an RCPL has a smaller number of peptides than a VCPL, it isinherently less likely to induce an autoimmune response. The sequenceschosen for the RCPL can be biased towards certain residues at certainpositions, analogous to the residue frequencies in a VCPL. An RCPL canalso be biased toward particular discrete sequences. In other words, ifan RCPL includes 100 distinct peptide sequences, those peptides can becombined in any arbitrary ratio. Despite the much smaller number ofsequences, the antigenic diversity of an RCPL can be as great as that ofVCPL from which the RCPL is created. In this way, a mixture of arelatively small number of chemical species (i.e. peptide sequences) canproduce an equally broad immune response as a mixture of a very largenumber of chemical species.

RCPLs are sets of homologous peptides evenly covering the diversevariants of hypervariable regions of infectious agents. RCPLs arecharacterized by smaller numbers of peptides in the libraries (comparedto VCPLs), which evenly cover the set of antigenic variants ofhypervariable regions of variable infectious agents, including potentialvariants. RCPLs are designed to mimic the antigenic diversity of mainvariable protective determinants of an infectious agent. RCPLs allowproduction of a wide range of antibodies and cytotoxic T-lymphocytes(CTLs), whose combined specificity covers the set of antigenic variantsof the variable infectious agent. Thus, RCPLs can induce a humoral andcellular response against the diversity of current and potentialvariants of variable infectious agents.

To select the specified number, N, of peptides in the RCPL, a largenumber (e.g. >1,000, >10,000, >100,000, >1,000,000, or >10,000,000) ofpeptides sequences are chosen at random from the whole diversity ofvariants of the mimicked hypervariable antigenically active region(e.g., a VCPL), accounting for the rates of occurrence of amino acids atparticular positions of the mimicked region. The set of randomly chosenpeptide sequences is arranged in the order of the relative rates oftheir occurrence in the whole diversity of the variants of the mimickedhypervariable antigenic region. The first 37-100% of the sequences fromthe set of randomly chosen peptides are used. The percentage used isdetermined by empirical considerations.

The peptide sequence having amino acids with the greatest rates ofoccurrence at each position is taken as the first sequence of the RCPL.Alternatively, any peptide of the set of variants of the mimickedhypervariable antigenic region may be used as the first, or reference,sequence.

Each subsequent peptide sequence added to the RCPL is chosen from theset of randomly chosen peptides such that the distance between the newsequence and all sequences previously added to the library is maximal orapproaches the maximum. The distance between two peptides, R_(ij), canbe determined with the use of the antigenic similarity matrix (see,e.g., Table 1). R_(ij) is calculated according to:$R_{ij} = {\sum\limits_{k = 1}^{l}{F( {i_{k},j_{k}} )}}$where i_(k) is the k^(th) residue of peptide i, j_(k) is the k^(th)residue of peptide j, and l is the number of amino acid residues in eachpeptide of the library. The function F can be described by an antigenicsimilarity matrix:F(i _(k) ,j _(k))=ASM(i _(k) ,j _(k))where the value of ASM(i_(k),j_(k)) is the matrix value for the pair ofresidues represented by i_(k) and j_(k).

For each peptide n from the randomly chosen peptides, the sum ofdistances S_(n) between the first N peptides selected (i.e., thosealready in the RCPL) and peptide n is calculated according to theformula: $S_{n} = {\sum\limits_{i = 1}^{N}R_{i\quad n}}$where R_(in) is calculated as desibed above. The peptide n that has thegreatest value of S_(n) is added to the library. The selection ofsequences from the large set of randomly chosen sequences continuesuntil the RCPL has the desired number of sequences.

The function F can alternatively be used simply to count the number ofsubstitutions between two peptides. In this case, the value ofF(i_(k),j_(k)) is 1 when i_(k) equals j_(k) (i.e. both peptides have thesame residue at position k), and F(i_(k), j_(k)) is 0 when i_(k) andj_(k) are different. When selected in this way, the sequences of theRCPL should evenly cover the whole set of variants of the mimickedhypervariable antigenically active region.

Each peptide included in the RCPL can be screened for autoimmunogenicproperties as described above. If a peptide sequence is determined tohave the potential to induce an autoimmune response, it can be excludedfrom the RCPL. Another candidate, virtually identical in the requiredproperties, is instead chosen as the next peptide in the RCPL. Thechoice algorithm is designed so that the each new peptide added to theRCPL is chosen from a number of sequences of equal value (i.e. of equaldistance from the previous peptide sequence in the library).

A sufficient number of peptides chosen as described here can evenlycover the whole diversity of the antigenic variants of hypervariableepitopes of variable infectious agents. The greater is the number ofpeptides making up an RCPL, the “denser” the immune response that it caninduce. Because the algorithm of choosing N peptides is incremental, thefirst N/2 peptides also evenly cover the whole diversity of thevariants, although less densely.

Libraries of representative chimeric peptides can be constructed ascandidate vaccines. Taken together, the peptide sequences in the librarycover the whole diversity of variants of hypervariable antigenicallyactive regions of a variable infectious agent. The sequences aredesigned to mimic the genetic diversity of main variable protectivedeterminants of infectious agents. Also, the mimicking peptides can bechimeric, because, although they mimic the heterogeneous set ofvariants, the sequences in the RCPL actually occur in few, if any, ofthe known and potential variants of epitope sequences.

In general, peptide libraries can be synthesized manually or by using anautomated synthesizer as described, e.g., in Lebl M. and Krchnak V.(1997) Solid-Phase Peptide Synthesis, Methods in Enzymology, 289,Academic Press, Minneapolis, Minn., which is incorporated by referencein its entirety. The synthesis can be a combinatorial synthesis, inwhich mixtures of amino acid are coupled to the growing peptide, and amixture of peptide sequences is formed. In a combinatorial synthesis,amino acids are added to a coupling reaction in proportions dictated bythe desired ratio of residues at each position in the peptide, adjustedfor the relative coupling rate for each amino acid. Alternatively, thelibrary can be produced by parallel synthesis, in which each sequence issynthesized separately, and the library can be a group of individuallypure peptide sequences. Such a library can be converted to a mixture bymixing the pure peptides.

Automated combinatorial synthesis can be preferable when a large libraryis to be produced. Split synthesis can be preferable when a smalllibrary of individual sequences (e.g. an RCPL) is to be produced.

The peptide libraries can be part of a multiple antigenic peptide (MAP).A multiple antigenic peptide includes a branched core from which severalpeptides extend. The branched core can be based on branching repeats oflysine. The MAP can include for example 4 or 8 peptide chains. The MAPcan include a hydrophobic group, such as an alkyl chain derived frompalmitic acid. The hydrophobic group can facilitate assembly of the MAPwith liposomes or virus-like particles.

The peptides can include the 20 common naturally occurring L-aminoacids. The peptides can optionally include corresponding D-amino acids,or mixtures of D and L-amino acids. Non-natural amino acids can likewisebe included in the peptides.

The peptide library can be produced by recombinant methods. A DNAconstruct encoding the peptides of the library can be introduced into ahost organism capable of protein expression from the construct. Suitablehost organisms can include bacteria such as E. coli, Lactobacillus, andBifida, and plants, such as tobacco.

The peptide libraries can be used in diagnostic applications, forexample to determine the presence of antibodies, or to determine CTLfunction. For example, the libraries may be used an antigens for anenzyme-linked immunosorbent assay (ELISA) to determine the presence ofantibodies in a subject sample. In a solid-phase assay such as an ELISA,the peptides of the library are immobilized on a substrate (for example,in a polystyrene well). The substrate is then exposed to a test sample(e.g., serum from a subject) under conditions that allow specificpeptide-antibody complex formation. Any unbound antibodies are thenwashed away. Antibodies associated with the substrate by virtue of aspecific peptide-antibody interaction are then detected. Detection caninclude exposing the antibodies to a secondary antibody specific forimmunoglobulins from the subject. The secondary antibody can beradiolabelled, or coupled to an enzyme such as horseradish peroxidase oralkaline phosphatase, that catalyzes the formation of a colored product.

The diagnostic peptide library can be a broad spectrum diagnosticreagent, or a subtype specific reagent. If the library was designed tobe antigenically similar to all known subtypes of a pathogen, then itcan be used to detect that a subject has been infected by the pathogen,regardless of what subtype the subject is infected with. If instead thelibrary was designed to be antigenically similar to only one subtype,then it can be used to detect if a subject was infected by the samesubtype. A group of subtype specific libraries can be used together todetermine which of several subtypes a subject was infected by.

Vaccines based on antigenic peptides are known. See, for example, U.S.Pat. Nos. 4,601,903, 4,599,230, and 4,599,231, each of which isincorporated by reference in its entirety. Vaccines may be prepared asinjectables, as liquid solutions or emulsions. The peptides may be mixedwith pharmaceutically acceptable excipients that are compatible with thepeptides. Excipients may include water, saline, dextrose, glycerol,ethanol, and combinations thereof. The vaccine may further containauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, or adjuvants to enhance the effectiveness of the vaccines.Methods of achieving adjuvant effect for the vaccine include the use ofagents such as aluminum hydroxide or phosphate (alum), commonly used as0.05 to 0.1 percent solution in phosphate buffered saline. Vaccines maybe administered parenterally, by injection subcutaneously orintramuscularly. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkylene glycols ortriglycerides. Oral formulations may include normally employedincipients such as, for example, pharmaceutical grades of saccharine,cellulose and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of micrograms of the peptides.Suitable regimes for initial administration and booster doses are alsovariable, but may include an initial administration followed bysubsequent administrations. The dosage of the vaccine may also depend onthe route of administration and will vary according to the size of thehost.

Nucleic acid molecules encoding the peptides of the library may also beused directly for immunization by administration of the nucleic acidmolecules directly, for example by injection, or by first constructing alive vector, such as Salmonella, BCG, adenovirus, poxvirus, vaccinia orpoliovirus, and administering the vector.

The vaccine can be a prophylactic vaccine that induces a protectiveimmune response in a subject, before the subject is exposed to thepathogen. In general, a prophylactic vaccine includes a broad-spectrumpeptide library. In some cases, a prophylactic vaccine can include asubtype-specific peptide library, for instance, when the subject to bevaccinated is likely to be exposed to only one subtype of the pathogen.The vaccine can be a therapeutic vaccine, designed to induce or enhancean immune response in a subject that is infected by the pathogen. If thesubject is known to be infected with a particular subtype of pathogen,the therapeutic vaccine can be one designed to induce an immune responseagainst that subtype.

EXAMPLES

Peptide libraries were designed based on each of the variable regionsV1-V5 of HIV gp120. The sequences used to generate the libraries werefrom HIV subtype A, B, C, D, F, G, or sequences from all these subtypeswere used together. A peptide library generated using sequences fromonly one subtype can be used as a subtype-specific vaccine, whereas alibrary generated from multiple subtypes can be used as a broad spectrumvaccine. Each library was initially designed to include fewer than200,000,000 sequences. From the initial library, smaller libraries weredesigned to include fewer than 500,000 sequences and fewer than 50,000sequences. Smaller libraries are designed by removing infrequentlyoccurring residues from the larger library. In Tables 3-37 below, theidentities and relative frequencies of residues are given. For example,in Table 3, residue X¹¹ can be S, N or G in the largest library; S or Nin the next smaller library, and S in the smallest library. The numbersrepresent the relative frequencies of each residue at that position. Inthe largest library, position X¹¹ is occupied by S, N or G in an 80:13:7ratio; in the next smaller library, X¹¹ is occupied by S or N in an80:13 ratio; and in the smallest library, every peptide has S at X¹¹.

Chimeric Peptide Libraries—V1 region of HIV gp120

Peptide libraries based on the V1 region of HIV gp120 from subtypes A,B, C, D, F, and G were designed. The libraries had the structure:

-   -   C—T—X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—E—X¹⁸—K—N

where X¹-X¹⁸ are defined according to Table 3. TABLE 3 HIV gp120 V1region, subtypes ABCDFG Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰X¹¹ X¹² X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸    50,000 D L K N T T N T N S S S G M EK G I 68 81 43 87 33 81 67 69 70 61 80 77 49 59 88 58 79 73 N T N T T NN S T E E M 32 22 26 33 30 20 15 33 15 16 21 27 N A N I 18 24 18 14 D 17   500,000 T N N K 13 13 13 12 200,000,000 W G N V A G E E G 12  8 10 1112  7  8 12 10 Y E V A I N  7  8 10  7  7  9 T  8

Peptide libraries based on the V1 region of HIV gp120 from subtype Awere designed. The libraries had the structure:

-   -   C—T—X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—E—X¹⁷—K—N

where X¹-X¹⁷ are defined according to Table 4. TABLE 4 HIV gp120 V1region, subtype A Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷    50,000 N V N V T S T S N T T E G D K E I 87 67 7076 65 53 62 42 45 34 57 32 37 29 62 69 82 D A T S N N N N A I T E M E G13 33 30 24 24 32 20 36 40 29 26 34 21 19 31 N N 22 20    500,000 S N SQ M 15 19 16 19 18 D 18 200,000,000 V V V V I V N G 11 15 10 14 13 13 1314 A A R Y T  8  8 13  8  9 D I  9  7 N  7

Peptide libraries based on the V1 region of HIV gp120 from subtype Bwere designed. The libraries had the structure:

-   -   C—T—X¹—X²—X³—N—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—E—X¹⁶—X¹⁷—E—X¹⁸—K—N

where X¹-X¹⁸ are defined according to Table 5. TABLE 5 HIV gp120 V1region, subtype B Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸    50,000 D L K A T N T N S S S G E I M K G I 8579 55 30 91 74 90 66 74 83  88 49 29 34 67 69 92 75 N T T T N S M T I GM 14 30 26 34 19 36 20 22 14 12 25 T N N G M 12 21 15 17 18 D T K 19 1718    500,000 N W N T 15 14 12 12 200,000,000 Y E N N A N I E E E E  711  9 10  7 8  9  8  7 11  8 G G K T  9 8  8  8

Peptide libraries based on the V1 region of HIV gp120 from subtype Cwere designed. The libraries had the structure:

-   -   C—X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—E—X¹⁸—K—N

where X¹-X¹⁸ are defined according to Table 6. TABLE 6 HIV gp120 V1region, subtype C Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸    50,000 T N A T R N G N V T Y N N T M E G I 7386 48 76 28 63 48 68 43 84 52 67 59 80 58 30 57 61 V N S T N T T V D I KE M 41 24 24 30 22 32 33 21 21 24 27 34 39 N S A N 20 22 24 26   500,000 R V S N T 19 18 18 20 18 200,000,000 V D T A D V N N T S T K 8 14 11 10  7  9 16  9 18 11 17  9 D G 15  9

Peptide libraries based on the V1 region of HIV gp120 from subtype Dwere designed. The libraries had the structure:

-   -   C—X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—T—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—E—X¹⁷—K—N

where X¹-X¹⁷ are defined according to Table 7. TABLE 7 HIV gp120 V1region, subtype D Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷    50,000 T D W K N N T T N N N T T M E G M 87 85 3845 47 73 35 39 44 55 47 45 75 32 64 71 91 A W G E A N G S T V D I G 2018 28 28 29 24 25 29 24 37 25 21 36 S K K E 25 17 18 19    500,000 N D AI 18 19 18 17 Y G 18 18 200,000,000 I N V T T K A N T V 13 15 17 16 1416 11 10 12  9 E N K 15 11  9 Y L T 11 11  9

Peptide libraries based on the V1 region of HIV gp120 from subtype Fwere designed. The libraries had the structure:

-   -   C—X¹—X²—X³—X⁴—X⁵—X⁶—T—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³T—L—K—X¹⁴—X¹⁵—X¹⁶—X¹⁷—E—X¹⁸—X¹⁹—X²⁰—N

where X¹-X²⁰ are defined according to Table 8. TABLE 8 HIV gp120 V1region, subtype F Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹ X²⁰    50,000 T N A T N A N D T I T N T E EP G A I Q 60 47 37 53 61 84 73 40 60 54 37 53 56 90 89 70 80 65 86 67 RD T I T S N N T N G D E K 28 32 25 47 27 27 27 27 46 32 35 31 35 33 N A25 31    500,000 T G Q E 16 20 18 20 200,000,000 A A I V S A D P I S S M12 11 13 12 13 13 12 13 10 11 12 14 S 11

Peptide libraries based on the V1 region of HIV gp120 from subtype Gwere designed. The libraries had the structure:

-   -   C—T—X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—N

where X¹-X¹⁷ are defined according to Table 9. TABLE 9 HIV gp120 V1region, subtype G Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷    50,000 N V T N N S T K N V T E K E E I K 77 88 6567 71 34 70 35 72 62 56 51 44 53 52 73 78 D N T C N T S E S E N R M 2335 29 26 16 25 22 17 19 24 31 29 27 N N A N 24 16 17 19    500,000 G C CG G 16 16 15 16 17 N N 14 15 200,000,000 A Y A E E V T 12 12 14 14 14 1112 K G T E 10 10 14 10 T 10

Chimeric Peptide Libraries—V2 region of HIV gp120

Peptide libraries based on the V2 region of HIV gp120 from subtypes A,B, C, D, F, and G were designed. The libraries had the structure:

-   -   S—X¹—N—X²—T—T—X³—X⁴—R—X⁵—K—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—L—F—Y—X¹²—L—D—V—V—X¹³—I—X¹⁴—X¹⁵—X¹⁶—X¹⁷—X¹⁸—X¹⁹—Y—X²⁰—L—X²¹—X²²—C

where X¹-X²² are defined according to Table 10. TABLE 10 HIV gp120 V2region, subtypes A, B, C, D, F, G Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸X⁹ X¹⁰ X¹¹   50,000 F I E I D V Q K E Y A 91 84 37 91 95 45 69 89 62 8979 M S K V S 16 32 40 33 21 N M 18 15    500,000 M K Q H  9 31 11 11200,000,000 Y V N Q  9  7  5  5 G  6 Library size < X¹² X¹³ X¹⁴ X¹⁵ X¹⁶X¹⁷ X¹⁸ X¹⁹ X²⁰ X²¹ X²²    50,000 K P D N D S T S R I S 93 73 63 78 5451 91 76 95 95 51 Q N D N N N N 27 22 22 32 49 10 49 S 15    500,000 N 9 200,000,000 S G E T T  7  8  8  5  5 K R  7  6

Peptide libraries based on the V2 region of HIV gp120 from subtype Awere designed. The libraries had the structure:

-   -   S—X¹—X²—X³—T—T—X⁴—L—R—D—K—K—X⁵—X⁶—V—X⁷—X⁸—L—F—Y—R—L—D—V—V—X⁹—I—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—X¹⁸—Y—R—L—I—X¹⁹—C

where X¹-X¹⁹ are defined according to Table 11. TABLE 11 HIV gp120 V2region, subtype A Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹    50,000 F N M E Q K Y S Q N E N N S S N SE N 63 91 58 92 62 90 79 83 59 61 68 62 71 86 82 69 59 60 96 Y I K H A PD S T N S N Q 37 42 38 15 17 35 20 15 20 18 18 32 18 K G D S 13 10 13 16   500,000 Q Q D 10 11  9 A 11 K  9 200,000,000 S V S K T K N E K S  9 8  6  6  6  8  9  4  6  4 D A K  4  5  4

Peptide libraries based on the V2 region of HIV gp120 from subtype Bwere designed. The libraries had the structure:

-   -   S—F—X¹—I—T—T—X²—X³—X⁴—X⁵—K—X⁶—X⁷—X⁸—X⁹—X¹⁰—A—X¹¹—F—X¹²—X¹³—L—D—V—V—X¹⁴—I—X¹⁵—X¹⁶—X¹⁷—X¹⁸—T—X¹⁹—Y—X²⁰—L—X²¹—X²²—C

where X¹-X²² are defined according to Table 12. TABLE 12 HIV gp120 V2region, subtype B Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹   50,000 N S I R D V Q K E Y L 94 53 85 94 85  68 80 95 93 95 95 N M NM K 28 15 8 24 20 G R 11  8 D  9    500,000 Q  7 200,000,000 K S G E H T6  6 7  5  5  5 Library size < X¹² X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹ X²⁰ X²¹X²²    50,000 Y K P D N D N S R I S 95 91 88 79 76 70 61 87  92 86  81 SQ G D N T T T N  9 13 11 18 22 39  8 7 19    500,000 S R R  7 7 7200,000,000 N K K N  5  5  7 6 N  5

Peptide libraries based on the V2 region of HIV gp120 from subtype Cwere designed. The libraries had the structure:

-   -   S—F—N—X¹—T—T—E—L—R—D—K—X²—X³—X⁴—X⁵—X⁶—A—L—F—Y—R—X⁷—D—I—V—X⁸—L—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—Y—X¹⁵—L—I—X¹⁶—C

where X¹-X¹⁶ are defined according to Table 13. TABLE 13 HIV gp 120 V2region, subtype C Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶    50,000 I K K K V Y L P N N N S S E R N 45 88  49 6153 60  94 87  63 71 56 89  71 76 90 96 A Q Q E H Q D S S G T I 21 42 2635 23  8 28 18 32 14 10 10 T T A K R 17 13 11  9  9 M 16    500,000 T DG K 7  7 7  7 200,000,000 Q E N P S K D N N N S 5  4 6  6 5  4  7 4  5 6  4 H S A  4 5  5 R 5

Peptide libraries based on the V2 region of HIV gp120 from subtype Dwere designed. The libraries had the structure:

-   -   S—F—N—I—T—T—X¹—V—X²—D—K—X³—X⁴—X⁵—X⁶—X⁷—A—L—X⁸—Y—R—X⁹—D—V—V—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—X¹⁸—Y—R—L—I—X¹⁹—C

where X¹-X¹⁹ are defined according to Table 14. TABLE 14 HIV gp120 V2region subtype D Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹    50,000 V R K K Q V Y F L Q I D N Q N S SS N 58 93 91 86  86 57 50 97 96 77 85 83 79 62 80 83 87  83 96 E Q K E HP M N T S T Q A N 38  9 10 34 35 23 15 11 12 22 20 13 8 14 Q K 10 13   500,000 I E G D  7 7  6  8 200,000,000 Q 7 A E K S Y P D G N D H  4 4  5  5  3  4  3  2 5  3  4 A K  4  2

Peptide libraries based on the V2 region of HIV gp120 from subtype Fwere designed. The libraries had the structure:

-   -   S—F—N—X¹—T—X²—E—V—R—D—K—X³—X⁴—K—X⁵—X⁶—A—L—F—Y—R—L—D—I—X⁷—X⁸—I—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—Y—R—L—X¹⁶—X¹⁷—X¹⁸

where X¹-X¹⁸ are defined according to Table 15. TABLE 15 HIV gp120region V2, subtype F Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹X¹² X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸    50,000 I T Q K V H V P N N S S N S T I NC 50 90 53 47 67 54 85 75 56 72 35 50 73 42 47 95 57 95 M K L E Q Q S ND S R E S 30 47 29 19 21 25 24 30 28 27 37 38 24 T Q D N 20 24 20 22   500,000 Y E G I H 15 15 15 15 14 200,000,000 M Q S K K G T G I 10 1411 10 14 10  5  5  5 G A N 10  7  5 M D  7  5

Peptide libraries based on the V2 region of HIV gp120 from subtype Gwere designed. The libraries had the structure:

-   -   S—F—N—X¹—T—X²—X³—X⁴—X⁵—D—K—X⁶—K—X⁷—E—Y—A—L—F—Y—R—X⁸—D—V—V—X⁹—I—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—Y—X¹⁶—L—X¹⁷—X¹⁸—C

where X¹-X¹⁸ are defined according to Table 16. TABLE 16 HIV gp120 V2region, subtype G Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸    50,000 I T E I R K T L P N D N N N N R I N94  95 97 97 93 80 51  95 91  60 85 49 54 63 66 87 95 97 Q Q K D G S S SS V  7 10 29  28 15 24 27 31 27 10 T A T G D G D  7 8  8 13 10  6  7 E DR 7  9  6    500,000 Q T Q T 5  5 5  5 200,000,000 M A A M E S R K I T H3  5  3  3  3 4  4  4  3  3  3 T 3

Chimeric Peptide Libraries—V3 Region of HIV gp120

Peptide libraries based on the V3 region of HIV gp120 from subtypes A,B, C, D, F, and G were designed. The libraries had the structure:

-   -   X¹C—X²—R—P—X³—N—N—T—R—X⁴—X⁵—X⁶—X⁷—X⁸—G—X⁹—G—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—T—G—X¹⁵—I—X¹⁶—G—X¹⁷—I—R—X¹⁸—A—X¹⁹—C—X²⁰

where X¹-X²⁰ are defined according to Table 17. TABLE 17 HIV gp120 V3region, subtypes A, B, C, D, F, G Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸X⁹ X¹⁰    50,000 N T N K S I H I P R 78  89 77 90  78 92  45 93 95 53 VI S G R Q 9 11 10 15 29 47 T P 8 12 S  8    500,000 G R N M  8  7  6  7200,000,000 Q T 5 4 E Y T M L 4  4 4 4  5 Library size < X¹¹ X¹² X¹³ X¹⁴X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹ X²⁰    50,000 A F Y A D 1 D Q H N 72 90 92  60 68 9686 85 85 95 T W T Q N K Y 23 10 40 14 14 15 15 A 11    500,000200,000,000 F 4 V H R T T  4 3  7  4  5

Peptide libraries based on the V3 region of HIV gp120 from subtype Awere designed. The libraries had the structure:

-   -   X¹—C—X²—R—P—X³—N—N—X⁴—R—X⁵—X⁶—V—X⁷—I—G—P—G—X⁸—X⁹—F—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—I—X¹⁵—G—X¹⁶—I—R—X¹⁷—A—X¹⁸—C—X¹⁹

where X¹-X¹⁹ are defined according to Table 18. A dash “—” indicates noresidue at that position. For example, if X¹² is represented by a dash,then no residue occupies X12, and the residue of X¹¹ is bound directlyto the residue of X¹³. TABLE 18 HIV gp120 V3 region, subtype A Librarysize < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹² X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸X¹⁹    50,000 N T N T K S R Q A Y A T G D I D Q H N 76 72 68 97 78 85 6693 56 95  95 91  89  79  92 84 76 76 87  T I G T G H R T — A T N K Y T19 28 23 19 11 34  7 41 6 12   8 16 22 24 7 S  9    500,000 T R D E  5 55 5 200,000,000 D K Q R V F A N E  5  3  3  2  3 3 2 4  2 N H — R  2 2 23 V 2

Peptide libraries based on the V3 region of HIV gp120 from subtype Bwere designed. The libraries had the structure:

-   -   X¹—C—X²—R—P—X³—N—N—T—R—K—X⁴—I—X⁵—X⁶—G—X⁷—G—X⁸—X⁹—X¹⁰—X¹¹—X¹²—T—X¹³—X¹⁴—I—X¹⁵—G—X¹⁶—I—R—X¹⁷—A—X¹⁸—C—X¹⁹

where X¹-X¹⁹ are defined according to Table 19. TABLE 19 HIV gp120 V3region, subtype B Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹    50,000 N T N S H I P R A F Y T G E I D QH N 94  91 86 75 58 89 94  85  90  80 91  59 94  62 97 88 89 91 95 I S GP M Q W A Q N K Y T  9 10 18 18 11 9 15 41 21 13 11  9  5 R N R  7 11  9T G 11  9    500,000 V F 5 4 200,000,000 T H 5 4 T G Y L G V E T 4  4  33 3  4 3  3 H W S R 2 3 3 3

Peptide libraries based on the V3 region of HIV gp120 from subtype Cwere designed. The library had the structure:

-   -   X¹—C—X²—R—P—X³—N—N—T—R—X⁴—X⁵—X⁶—X⁷—I—G—P—G—Q—X⁸—F—X⁹—X¹⁰—T—X¹¹—X¹²—I—X¹³—G—X¹⁴—I—R—X¹⁵—A—X¹⁶—C—X¹⁷

where X¹-X¹⁷ are defined according to Table 20. TABLE 20 HIV gp120 V3region, subtype C Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷    50,000 V T N K S I R T Y A G D I D Q H N 57  91 77 88  98 76 97 82 96 98 85  77  98 87 90  82 92  N I G E M G A F N G NK Y T 26  6 12 9  24  3 16  4 11  20  13 6 17 4 E S 9 10 T 4    500,000A Q T E I 3 3  2 2 2 200,000,000 M H G V T D N L N D 2  1  2  2  2 2 1 1 1 1 R K S H H 2 1 1 1 1 T I 1 1

Peptide libraries based on the V3 region of HIV gp120 from subtype Dwere designed. The libraries had the structure:

-   -   X¹—C—X²—R—P—X³—X⁴—X⁵—X⁶—R—X⁷—X⁸—X⁹—X¹⁰—I—G—X¹¹—G—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—T—X¹⁷—E—X¹⁸—G—X¹⁹—I—X²⁰—X²¹—A—X²²—C—X²³

where X¹-X²³ are defined according to Table 21. TABLE 21 HIV gp120 V3region, subtype D Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²   50,000 N T Y N N T Q S T H P Q 78 83 74 87 89 72 71 37 74 61 64 70 TI N R R I P L R 22 17 26 29 35 26 19 16 30 G 28    500,000 K I S 13 1611 K 12 200,000,000 K S Q 11 10  8 R  9 Library size < X¹³ X¹⁴ X¹⁵ X¹⁶X¹⁷ X¹⁸ X¹⁹ X²⁰ X²¹ X²² X²³    50,000 A L Y T I I D R Q H N 92 80 80 9282 83 84 92 84 67 82 Y F K N K Y 20 20 18 16 16 33    500,000200,000,000 T A T G T  8  8 10  8 11 K K  8  8

Peptide libraries based on the V3 region of HIV gp120 from subtype Fwere designed. The libraries had the structure:

-   -   X¹—C—T—R—P—X²—N—N—X³—R—K—X⁴—I—X⁵—L—P—G—P—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—I—X¹⁴—G—X¹⁵—I—R—X¹⁶—A—X¹⁷—C—X¹⁸

where X¹-X¹⁸ are defined according to Table 22. TABLE 22 HIV gp120 V3region, subtype F Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸    50,000 N N T S H Q A F Y A T G D I D K H N 9396 97 91  73  69 87 97 92  73 95 92  87  96 91 92 86 83 T G R R V T A DA T N Q Y I  7 6 10  28 10 27  5 5 8  4  9  8 14 11 Q N 6 5    500,000 SP H  4 4 4 Y F 4 4 200,000,000 I R S H T I S D  3 3 3  3  3  3 3  3 K  3

Peptide libraries based on the V3 region of HIV gp120 from subtype Gwere designed. The libraries had the structure:

-   -   X¹—C—X²—R—P—X³—N—N—T—R—K—S—X⁴—X⁵—X⁶—G—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—T—X¹⁴—I—X¹⁵—G—X¹⁶—I—R—X¹⁷—A—X¹⁸—C—X¹⁹

where X¹-X¹⁹ are defined according to Table 23. TABLE 23 HIV gp120 V3region, subtype G Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹    50,000 N T N I T F P G Q A F Y A G I D QH N 44 90 88 98 32 66 93  98 86  76 93  98 95 91  98 76 94  83 96  I I SR I R T I T D N Y 19 10 12 25 34 7 17 5  5 5 24 15 T H V 15 22  7 M P 1215 R N  9  5    500,000 T H S K 5 5 4 2 200,000,000 K L R G S H T L F T 2 2  2 2 2  2  2 2  2 2 K 2 P 2

Chimeric Peptide Libraries—V4 region of HIV gp120

Peptide libraries based on the V4 region of HIV gp120 from subtypes A,B, C, D, F and G were designed. The libraries had the structure:

-   -   C—N—T—T—X¹—L—F—N—S—T—W—X²—X³—X⁴—X⁵—W—X⁶—T—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—X¹⁷—I—X¹⁸—L—X¹⁹—C

where X¹-X¹⁹ are defined according to Table 24. TABLE 24 HIV gp120 V4region, subtypes ABCDFG Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰X¹¹ X¹² X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹    50,000 Q N N S T N E G S N N T EG N D T T P 67 52 78 61 90 37 58 70 79 86 59 90 67 78 70 63 65 85 84 K FN S E L S S S N N I Q 33 18 22 33 18 21 24 22 30 13 25 15 16 T G D 16 1718 V T S D 14 15 12 14    500,000 N 14 K 13 200,000,000 S A D N K G I 1110 11 10 12 12  9 G V T S 10  9 11 12 G N  8 10

Peptide libraries based on the V4 region of HIV gp120 from subtype Awere designed. The libraries had the structure:

-   -   C—X¹—T—S—X²—L—F—N—X³—T—W—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—T—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—I—X¹⁷—L—X¹⁸—C

where X¹-X¹⁸ are defined according to Table 25. TABLE 25 HIV gp120 V4region, subtype A Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸    50,000 N G S T N I Q E S N S E S N D T T P 8756  90 43 62 26 48 58 85 91  85  60 80 85  72 66 91 66 D N S T T S L K LG N Q 22  32 20 25 41 17 15 17 20 28 28 34 E M G G 25 23 14 16 G 14   500,000 D G A N E V 13 10 10 12 10 10 200,000,000 S D D D N S K I 8 8 9 9  7 9  6  9 K N D 7 8 6 N I 7 7

Peptide libraries based on the V4 region of HIV gp120 from subtype Bwere designed. The libraries had the structure:

-   -   C—N—T—T—X¹——F—N—S—T—W—X²—X³—X⁴—X⁵—X⁶—X⁷—T—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—G—X¹⁵—X¹⁶—X¹⁷—I—X¹⁸—L—X¹⁹—C

where X¹-X¹⁹ are defined according to Table 26. TABLE 26 HIV gp120region V4, subtype B Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹X¹² X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷ X¹⁸ X¹⁹    50,000 Q N N S T W N E G S N N T E ND T T P 64 55 89 62 90 90 43 61 74 79 85 59 89 69 72 65 63 83 86 K T N ST E L D K S N N I 27 16 20 34 15 17 21 16 12 28 13 27 17 F G K S 16 1715 16    500,000 V D D S Q 13 13 15 11 14 200,000,000 P G A G V N R G NT G I  9 11 10 10 10  9 10  9 11 10 11  9 N  9

Peptide libraries based on the V4 region of HIV gp120 from subtype Cwere designed. The libraries had the structure:

-   -   C—N—T—S—X¹—L—F—N—X²—T—Y—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—X¹⁵—X¹⁶—L—X¹⁷—C

where X¹-X¹⁷ are defined according to Table 27. TABLE 27 HIV gp 120 V4region, subtype C Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶ X¹⁷    50,000 K S N N G T Y N S T E S N S T T P 36 65 4758 63 91 50 78 58 79 45 59 78 70 71 92 77 G G M S S S G N S D S N Q 2435 27 25 18 20 19 21 28 15 22 16 23 N S P N 20 18 17 15 F A I 16 16 13   500,000 N E 14 15 200,000,000 S R D L I N G N I 10  8 10  9 11 13 1211  8 D N T D V  9  8 11 10  9 G  7

Peptide libraries based on the V4 region of HIV gp120 from subtype Dwere designed. The libraries had the structure:

-   -   C—X¹—T—S—X²—L—F—N—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—I—X¹⁵—C

where X¹-X¹⁵ are defined according to Table 28 TABLE 28 HIV gp 120 V4region, subtype D Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵    50,000 N K S T W D N N T G D N I T P 94 62 83  92  85 4047  32 78  43 60 74 70 54 67 G N T G W N T M N S 26 26 42  26 25 17 2630 24 21 N S S N S I Q 13 23 23 17 12 13 11 E T 12 10    500,000 L K N GK 11  9 8  7  9 L 8 200,000,000 D R E Y G I K E D V  6 7 5  4  4 6  7 5 5  4 N V I D 6 3  3 2 G K 5 2

Peptide libraries based on the V4 region of HIV gp120 from subtype Fwere designed. The libraries had the structure:

-   -   X¹—X²—T—X³—X⁴—L—F—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—I—X¹⁴—L—X¹⁵—C

where X¹-X¹⁵ are defined according to Table 29. TABLE 29 HIV gp120 V4region, subtype F Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵    50,000 C N S G N D T N D T N G T T P 95 91 91  53 85  4492  81  46  84 86  53 81  82 89  D E T N T N A T D N I L  9 20 9 31 628  10 6 32 8 18 8 K S S S I 10 17 10   9 6 Q  8    500,000 D I K A G NP 6  6 6  5 5  6 5 200,000,000 Y A S H A E M N K Q  5 3  3  3 4 5 4  3 43 V I K I D 4 5 3  3 4 A I 3 3

Peptide libraries based on the V4 region of HIV gp120 from subtype Gwere designed. The libraries had the structure:

-   -   C—N—T—S—X¹—L—F—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²—X¹³—X¹⁴—I—X¹⁵—L—X¹⁶—C

where X¹-X¹⁶ are defined according to Table 30. TABLE 30 HIV gp120 V4region, subtype G Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²X¹³ X¹⁴ X¹⁵ X¹⁶    50,000 G N N S N S N T S N N N T T T P 54 86  54 88 63 57 61 39 88  35 61 74 45 78 87  95 K S S D S I E S D D N 26 25 16 2234 23 29 27 15 34 15 E N N K E G 21 20 14 14 12 21 E T 12 14    500,000E K A S 11 12  8 11 Q D 10  9 200,000,000 R N D A A I I Q 8 6  5  7 7  67  5 T E R N M 6 6  4 6 6

Chimeric Peptide Libraries—V5 region of HIV gp120

Peptide libraries based on the V5 region of HIV gp120 from subtypes A,B, C, D, F, and G were designed. The libraries had the structure:

-   -   X¹—X²—X³—X⁴—X⁵—X⁶—E—X⁷—F—R—P—X⁸

where X¹-X⁸ are defined according to Table 31. TABLE 31 HIV gp120 V5region, subtypes ABCDFG Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸  50,000 NS N N E T I G 75  42 42 68  35 75  53 99 D N T S T N T 9 25 36 18  2320  47 G E D E N P 6 16 13 6 18 2 T G K G G I 5 10  5 5 13 2 K K G K K 3 5  4 3 10 P I 2  1 500,000 M E E  1 1  1 H  1

Peptide libraries based on the V5 region of HIV gp120 from subtype Awere designed. The libraries had the structure:

-   -   X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—R—P—X¹⁰

where X¹-X¹⁰ are defined according to Table 32. TABLE 32 HIV gp120 V5region, subtype A Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰   50,000 G N N S T N E T F G 31 60  76  76  57  61  99 85 99 78 N T S NE T I I 24 22  17  16  16  27  15 16 D G D R N E T 24 6 3 4 10  7  4 S DA P  8 5 9 3 K I I  6 4 4 V K  6 3    500,000 G K K Q S R 2 2 2  1  1  2M 1 200,000,000 R E P 1 1 1 G F 1 1 I 1 H 1

Peptide libraries based on the V5 region of HIV gp120 from subtype Bwere designed. The libraries had the structure:

-   -   X¹—X²—X³—X⁴—X⁵—X⁶—E—X⁷—F—R—P—G

where X¹-X⁷ are defined according to Table 33. TABLE 33 HIV gp120 V5region, subtype B Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ 50,000 N S N N E TI 78  44 40 69  37 77 57 D N T S T N T 9 25 37 16  21 19 43 G E D G N P4 16 15 6 18  2 T G K E G I 4  9  5 6 13  2 K K G K K 3  6  4 3 10 P M M2  1  2

Peptide libraries based on the V5 region of HIV gp120 from subtype Cwere designed. The libraries had the structure:

-   -   X¹—X²—X³—X⁴—X⁵—E—X⁶—X⁷—X⁸—X⁹—X¹⁰

where X¹-X¹⁰ are defined according to Table 34. TABLE 34 HIV gp120 V5region, subtype C Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰   50,000 E T N D T T F R P G 45 44 81 28 85  55 99 98  99 89  N N T N NI E 18 16 13 26 6 42 5 T D D T E K I 12 13  4 85 5  2 2 G G K G Q 11 13 6 2 2 K K G I T  7  7  9 2 2 L I  5  5    500,000 P P G I Q T  2  2  2 2 1  1 200,000,000 N I S  1  1 1

Peptide libraries based on the V5 region of HIV gp120 from subtype Dwere designed. The libraries had the structure:

-   -   X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—E—X⁸—X⁹—R—X¹⁰-X¹¹

where X¹-X¹¹ are defined according to Table 35. TABLE 35 HIV gp120 V5region, subtype D Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹   50,000 N N N S N Q N T F P G 58  92  44  70  52  50 78 94 97 98 99 TG S N S E S I I 13  3 40  25  32  22 18  6  3 A D D R H 13  3 6 7 12 K GG S 9 6 4 10 E H P 3 3  3 H 3    500,000 I G D R H T 2 2 2  2  2  2 H RR 2 2  2 200,000,000 Y H D G Q 1 1 1  1  1 K 1

Peptide libraries based on the V5 region of HIV gp120 from subtype Fwere designed. The libraries had the structure:

-   -   X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—F—R—P—X⁸

where X¹-X⁸ are defined according to Table 36. TABLE 36 HIV gp120 V5region, subtype F Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ 50,000 N N N TN E T G 43 65  46  51 60 97 83 80 L D S K S Q I E 30 14  35  14 29  3 1710 E G G D D I 16 7 7 14  7  7 S K E N I Q  8 7 6 14  4  3 K T K G  4 76  7

Peptide libraries based on the V5 region of HIV gp120 from subtype Gwere designed. The libraries had the structure:

-   -   X¹—X²—X³—X⁴—X⁵—X⁶—X⁷—X⁸—X⁹—X¹⁰—X¹¹—X¹²

where X¹-X¹² are defined according to Table 37. TABLE 37 HIV gp120 V5region, subtype G Library size < X¹ X² X³ X⁴ X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ X¹²   50,000 N N N S T N E T F R P G 40 78  75 53  85  63  98  82 91  93 87  88  A T T N G T I I A R 18 9 10 35  7 25  17 5 7 8 T A D E E T 16 810 4 6 4 D A 15 4 R  9    500,000 A E G N E  3 3 4 2 4 T S 2 2200,000,000 Y K K I K K N D G D E  2 2  2 2 2 1 1  1 1 1 1 D K N F S K W2 1 2 1 1 1 1 P H F Y 1 1 1 1 V 1

Synthesis and Antigenic Properties of a VCPL

A peptide library mimicking the naturally occurring diversity ofsequences in the main hypervariable V3 region of the HIV-1 (subtype B)gp120 envelope protein was designed. The identity and frequency of theamino acids in the library were chosen based on sequence alignments andantigenic similarity. HIV-1 sequences are available from the HIVSequence Database (hiv-web.lanl.gov). The peptide library had thestructure:

-   -   N—N—N—T—R—K—X¹—I—X²—X³—X⁴—X⁵—G—X⁶—X⁷—X⁸—Y—X⁹—T—G—X¹⁰I—I—G—X¹¹—R—Q

where X¹- X¹¹ were defined according to Table 38. TABLE 38 X¹ X² X³ X⁴X⁵ X⁶ X⁷ X⁸ X⁹ X¹⁰ X¹¹ S H I G P R A F T E D 75 60 82 97 95 77 v 93 7658 39 88 G P M A W Q T W A D N 18 18 12  3  5 9  7 15 42 28 12 R N K L Q 7 11 8  9 24 S S R 11 3  9 G 3

The peptides of the library were synthesized on Applied Biosystems 430Aand Vega Coupler C250 automatic synthesizers with the use of the Boc/Bzlstrategy of peptide chain elongation. A styrene copolymer with 1%divinylbenzene was used as the polymer support, and aphenacylamidomethyl (PAM) group served as the anchor group (Sparrow, J.T. J. Org. Chem. 1976, 41:1350-1353, which is incorporated by referencein its entirety). Condensation was done with 1-hydroxybenzotriazoleesters prepared directly before use by mixing a 3-fold molar excessrelative to the polymers of the Boc product of amino acid,1-hydroxybenzotriazole and diisopropylcarbodiimide. The completeness ofthe condensation was monitored (Kaiser, E. 398, which is incorporated byreference in its entirety). In the event of a positive ninhydrin testafter neutralization of the peptidyl polymer, condensation was repeated.If the test was negative, then the temporary Boc protective groups wereremoved with undiluted trifluoroacetic acid, and neutralization wasperformed by adding N,N′-diisopropylethylamine directly to the reactionmixture (Schnolzer, M. et al. Int. J. Pept. Prot Res. 1992, 40: 180-183,which is incorporated by reference in its entirety). After removal ofthe Boc groups the following amino acid residue was attached. Permanentprotective groups used for side chains were: 2-chlorobenzyloxycarbonylgroups for lysine, dichlorobenzyl for tyrosine, the corresponding benzylesters for aspartic acid, glutamic acid, serine and threonine, formylfor tryptophan, tosyl for arginine, and methionine was introduced in theform of the corresponding sulfoxide. Upon completion of assembly of theprotective polypeptide chain, the end products were deblocked with aone-time cleavage from the polymer with the aid of anhydrous hydrogenfluoride in the presence of scavengers (Tam, J. P. et al. J. Am. Chem.Soc. 1983, 105: 6442-6444, which is incorporated by reference in itsentirety).

During the synthesis of peptide libraries a mixture of protected aminoacid residues was added in the condensation stage in variable positionsrelative to the peptidyl polymer in a certain quantitative relationship,which provided a given incorporation of different amino acid residues tothe growing polypeptide chain. Because different amino acids havedifferent rates of amino group acylation, a number of model compoundswere synthesized and reaction rate coefficients were derived. The Bocproducts of the amino acids which were introduced into the mixture, wereused in ratios adjusted for the reaction rate coefficients, to providepeptide products having the desired ratios (as shown in Table 38) in thevariable positions.

The peptide library was purified by gel permeation chromatography usingSepharose G-50 and 50% acetic acid as the eluent. The synthesizedcompounds were characterized by analytical single-photonhigh-performance liquid chromatography (Gilson chromatograph, France;Xterra RP18 column, 125 Å, 3.9×150 mm, 5 μm, flow rate 1 mL/min, eluent0.1% trifluoroacetic acid, 10-40% and 20-40% acetonitrile gradient in 16min for pentarphin and cyclopentarphin, respectively), and by amino acidanalysis (hydrolysis 6N HCl, 24 hours, 110° C.; LKB amino acid analyzer,Alpha Plus, Sweden). The purity of the end products according toanalytic HPLC was not less than 95%. The amino acid composition ofpeptide hydrolysates (6N HCl, 120° C., 24 hours, LKB 4151 analyzer,Alpha Plus) corresponded to the theoretical.

BALB/c line mice ages 5-6 weeks were immunized 3 times intraperitoneallywith the peptide library diluted in a phosphate buffer solution withComplete Freund's Adjuvant (CFA). The mice were administered 0.2 mLpeptide solution in a dose of 50 μg/head. The second and thirdimmunization were conducted 2 and 4 weeks after the first immunization,respectively. A control group of animals were administered a peptidesolution mimicking the antigenic portion of protein E of the tickencephalitis virus (peptide sequence: KRDQSDRGWGNHAGLFGKGSIVT) accordingto the same scheme and in the same doses. Ten days after the lastimmunization a blood sample was taken from the retroorbital vein of theanimals for determination of the titer of specific antibodies in theserum.

The following antigens were immobilized in separate wells of microtiterplates for immunoenzyme analysis (Medpolimer, Moscow): thechimeric-peptide library, and each of 15 synthetic peptides representingthe amino acid sequences of the V3 region of gp120 from different HIV-1subtypes. The 15 synthetic peptides represent the consensus sequences ofappropriate subtypes. Where several peptides are present from onesubtype, they represent different variants.

The antigens were dissolved in 0.05 M carbonate-bicarbonate buffersolution, pH 9.6 to a final concentration of 10 μg/mL, placed in eachwell of the microtiter plate in 0.1 mL amounts and incubated overnightat a temperature of 37° C. The wells were washed 3 times with phosphatebuffer solution with Tween 20, and non-specific binding was blocked witha 0.5% solution of bovine serum albumin, fraction 5 (Sigma). Mouse bloodsera samples were serially diluted two-fold in a Tris-HCl buffersolution, and placed in 0.1 mL amounts into the wells with immobilizedantigen and incubated at 37° C. for 30 min. After incubation the wellswere washed 5 times with a phosphate buffer solution. Theantigen-antibody complexes were determined by adding 0.1 mL quantitiesof horseradish peroxidase-conjugated goat antibodies against mouse IgG(Sigma) in phosphate buffer solution. Following incubation at 37° C. for30 min, a substrate mixture containing orthophenylene diamine was added.The enzyme reaction was halted by the addition of a sulfuric acidsolution, and the optical density at 492 nm was measured using aMultiscan EX microplate photometer. The results of titer determinationof the antigen-specific antibodies in the mouse blood sera are presentedin Table 39.

Immunization of mice with the chimeric-peptide library caused formationof serum antibodies specific both for the chimeric-peptide libraryitself (the immunizing antigens) and for peptides forming antigenicdeterminants in the V3 region of some known HIV-1 subtypes. Inparticular, the antibody titers for subtype B peptides were higher thanfor other subtypes, as would be expected for a peptide library design tomimic the diversity of subtype B and not other subtypes. The chimericpeptide library was immunogenic, and induced formation of antibodiesthat interact with antigenic determinants of a broad spectrum of HIV-1variants. TABLE 39 Immobilized antigen Mean titer of (peptidescorresponding to specific region V3 gp120 HIV-1, subtype) antibodiesChimeric-peptide library  1:1600 Peptide No 1, subtype A 1:400 PeptideNo 2, subtype B  1:3200 Peptide No 3, subtype B  1:1600 Peptide No 4,subtype B 1:800 Peptide No 5, subtype B  1:1600 Peptide No 6, subtype B 1:1600 Peptide No 7, subtype C 1:400 Peptide No 8, subtype D 1:400Peptide No 9, subtype E 1:400 Peptide No 10, subtype E 1:200 Peptide No11, subtype F 1:100 Peptide No 12, subtype G <1:100   Peptide No 13,subtype H <1:100   Peptide No 14, subtype I 1:100 Peptide No 15, subtypeJ 1:100 Peptide of protein E of <1:100   the tick encephalitis virus(control)

The peptide library described above was tested as a diagnostic reagentin solid-phase immunoenzyme analysis (i.e., ELISA) of serum samples from50 HIV-infected patients and 30 healthy donors for the purpose ofdetermining specific antibodies. Preliminarily sera of HIV-infectedpatients were analyzed with the aid of a number of commercial testsystems. In addition, the peptide library was assessed for its abilityto detect antibodies to HIV-1 with the aid of standard sera panelsanti-HIV-1 series 010 and series 007.

The chimeric-peptide library was dissolved in 0.05 Mcarbonate-bicarbonate buffer solution, pH 9.6 to a final concentrationof 1.5 micrograms/mL. The solutions were added in 0.1 mL quantities tothe wells of the microtiter plates and incubated at room temperatureovernight, to immobilize the peptides in the wells. Then the wells werewashed 3-5 times with a solution of FSB-T (phosphate-salt buffersolution containing Tween 20). Henceforth the method completelycorresponds to the instructions for the use of the immunoenzyme testsystem for detecting antibodies to HIV-1 produced by DGUEPP“Vektor-BiAlgam” (Kol'tsovo, Novosibirsk Oblast).

Blood sera samples were diluted 20 fold in an FSB-T solution containing0.2% casein and were placed in wells of the microtiter plate in 0.1 mLamounts and incubated at 37° C. for 30 min, to bind antibodies to theimmobilized peptides. Then the wells were washed 5-7 times with FSB-Tsolution. To the wells were added 0.1 mL amounts of a monoclonalantibody to IgG conjugated to horseradish peroxidase in a dilution of1:20 in FSB-T containing 0.2% casein. The microtiter plate was incubatedat 37° C. for 30 min, then washed 5-7 times with an FSB-T solution.Next, 0.1 mL amounts of a solution of substrate mixture of 0.05% TMB and0.005% hydrogen peroxide in 0.05 M phosphate-citrate buffer solution, pH5.0 was added to the wells. The microtiter plate was kept at roomtemperature (18-22° C.) for 15-20 min in the dark. The reaction wasstopped by addition of 0.05 mL amounts of 2 M sulfuric acid solution tothe wells. Calculation of the results is accomplished by measurement ofthe optical density of the samples on a Multiscan-type automaticspectrophotometer at a wavelength of 450 nm.

As a control for ruling out non-specific results in setting up thereaction a substrate mixture (BCP+substrate) is used, along with acontrol conjugate (BCP+conjugate+substrate), and a control serum whichdoes not contain antibodies to HIV according to data from testing inRussian and foreign third generation screening test systems.

The test sera are judged to have antibodies to HIV-1 if the values ofthe optical density (OD) of the corresponding investigative solution inthe wells with the investigative sera exceed the critical level ODcrit,calculated according to the formula:ODcrit=OD(C′)_(mean)+0.2where OD(C′)_(mean) is the mean optical density for negative controlsera.

The results of the study of the anti-HIV-1 standard panel sera series010 and 007 with the use of the chimeric-peptide library showed an 81%sensitivity and 93% specificity. In addition, a study was undertaken ofthe effectiveness of the chimeric-peptide library for detection ofspecific antibodies to HIV-1 in blood sera samples with the use of 50sera from HIV-infected patients and 30 sera from healthy donors. Theresults of the tests showed more than 80% detection of positive samplescontaining specific antibodies to HIV-1 antigens on the basis of resultswith commercial third generation test systems.

To test the immunogenicity of the chimeric peptide library in differentantigen forms, the library was synthesized as linear peptides, amultiple antigen peptide of 4 linear peptides (MAP4), or multipleantigen peptide of 8 linear peptides (MAP8). Rabbits were immunizedsubcutaneously with 300 micrograms of the constructions described abovemixed with either complete Freund's adjuvant (first immunization) orincomplete Freund's adjuvant (second and third immunizations) at 12 dayintervals. At day 12 after the third immunization, the rabbits were bledand all blood sera were tested by ELISA against the linear, MAP4 andMAP8 antigens. Table 40 illustrates that immunization of rabbits withthe HIV-1 V3 library in different antigen forms induces a strong humoralimmune response to the highly variable V3 loop sequences from chimericpeptide library. TABLE 40 Antibody titers in ELISA for different antigenforms Immunizing dose linear MAP4 MAP8 linear 5,120 — — MAP4 81,920163,840 163,840 MAP8 10,240 81,920 81,920

The immunogenicity of the MAP4 chimeric peptide library in differentformulations was tested. The library was formulated in Freund'sadjuvant, artificial virus like particles (VLP) or in phosphate bufferedsolution (PBS). In addition, the MAP4 construction was modified withpalmitic acid (MAP4-P) and formulated in liposomes or microemulsion. Theliposomes included 100 micrograms of MAP4-P and 250 micrograms of dsRNA,and the microemulsion included 100 micrograms of MAP4-P and 100micrograms of dsRNA.

The VLPs contain a DNA molecule covered with polypeptides carrying theMAP4 peptide library. The target polypeptides are exposed on the surfaceof the particle and are attached to DNA via spermidine-polygluquineconjugates. The dimensions of the particle will allow the targetantigens to conjugate on the surface in copy numbers ranging from one toseveral hundred. The core of the VLP can contain DNA segments as largeas 10,000 bp. See, for example, Lebedev, L. R. et al. (2000) Moleculargenetics, microbiology and virology (Russian)(3), 36-40; Lebedev, L. R.,et al. (2000). Molecular Biology (Russian) 34(3), 480-485; and Lebedev,L. R. et al. (2001) Biotechnology (Russian)(1), 3-12; and Sizov, A. A.et al. (2001) Biotechnology (Russian)(1), 13-18, each of which isincorporated by reference in its entirety.

The liposomes forms were prepared with the MAP4-P library. The palmiticacid modification increases the lipophilicity of the peptides. Theliposomal compositions of the peptide were made bysolubilization-injection. The size of the bulk of liposomes was within200 nm.

The microemulsions were also made with the MAP4-P peptide construct andcontained (in 1 mL) 100 micrograms of peptide, 250 micrograms ofridostin, and 800 micrograms of the commercial (ICN) cationic amphiphiledimethyldioctadecyl ammonium bromide (DDAB).

Mice (five mice in each group) were immunized intraperitoneally with theconstructions described above three times at 12 days intervals. The datain Table 41 indicate the high diversity of individual immune response inconventional mice, but nevertheless strong humoral immune response wasdetected for all systems after 3^(rd) immunization, except for thepeptide library solution in PBS. The antigen formulation of MAP4-P inliposomes induced high antibody levels after the second immunization.TABLE 41 AB titers in mouse serum in ELISA for Antigen Total MAP4 insolid phase AB Titer, AB Antigen Delivery Dose Im. Mouse # AB titer,Stand. Titer, Form System μg No. 1 2 3 4 5 Average Deviation I95 MAP4PBS 40 2^(nd) 0 0 0 0 — 0 0 60 3^(rd) 0 0 0 0 — 0 0 MAP4 Freund's 402^(nd) 0 3200 1600 1600  12800 3840 5135 4501 adjuvant 60 3^(rd) 5120012800 102400 25600 204800 79360 78070 68430 MAP4-P Liposome 40 2^(nd) 051200 51200 25600 — 32000 24510 24019 60 3^(rd) 51200 102400 51200102400 — 76800 29560 28969 MAP4-P Microemulsion 40 2^(nd) 0 0 6400 25600— 8000 12115 11872 60 3^(rd) 0 0 204800 51200 — 64000 96920 94980 MAP4-PVLP 100 2^(nd) 12800 12800 6400 — — 10667 3695 4181 150 3^(rd) 51200204800 25600 — — 93867 96920 109673

To determine the polarization of the immune response of mice immunizedwith the MAP4 library in different delivery systems described above, onday 10 after the 3^(rd) immunization, splenocytes were distinguished.The splenocytes were stimulated in vitro with the library of linearpeptides. A number of splenocytes produced IFN-gamma or IL-4 cytokines,markers of Th1 or Th2 immune response, respectively, as measured byELISPOT. The number of cells that produced one of two cytokines wasnoted, after stimulation or without stimulation (Table 42). The resultspresented in Table 42 show that immunization of mice with a solution ofMAP4 peptide library with Freund's Adjuvant does not result in a majordifference (significance level 0.05) in the number of stimulated cellsfrom the control measurement (without stimulation), which confirms theabsence of induction of the T-helper response with immunization byantigens in a mixture with Freund's Adjuvant. The highest number ofcells producing IFN-gamma after stimulation in vitro using the linearpeptide library was noted in mice immunized with a microemulsioncontaining the MAP4 library. The immunization using VLP resulted in theformation of a less pronounced immune response of the Th1 type.Comparison of the number of cells producing IL4 and IFN-gamma revealsthat immunization of mice with the MAP4 library in VLPs results in abalanced T-helper response, i.e. the number of cells producing IFN-gammaand IL-4 does not significantly differ. On the other hand, immunizationof mice with the same antigens in a microemulsion results in asignificant predominance of the Th1 type immune response afterstimulation with the library of linear peptides. Thus, the above exampleshows the possibility of inducing a strong T-helper response using theMAP4 library of chimeric peptides. TABLE 42 Antigen Splenocytes AverageI95 of delivery stimulated in Cell sample ## No. of Standard cell systemvitro? 1 2 3 4 5 6 cells Deviation number Number of cells producingIFN-gamma Determined for 500,000 splenocytes in ELISPOT VLP Yes 59 99272 — 55 29 102.80 97.84 85.76 No 2 5 1 — 3 5 3.20 1.79 1.57 CFA/IFA Yes6 3 33 37 12 10 16.83 14.47 11.58 No 1 0 7 6 1 1 2.67 3.01 2.41 MicroYes 165 145 525 450 600 600 414.17 208.41 166.76 emulsion No 4 0 1 2 3 11.83 1.47 1.18 Number of cells producing IL-4. Determined for 500,000splenocytes in ELISPOT VLP Yes 21 36 420 490 61 43 178.50 215.70 172.59No 11 8 9 13 3 0 7.33 4.93 3.94 CFA/IFA Yes 47 24 39 54 2 5 28.50 21.8117.45 No 27 33 26 31 4 4 20.83 13.29 10.63 Micro Yes 56 42 159 135 79 7991.67 45.76 36.62 emulsion No 11 4 17 16 3 2 8.83 6.74 5.39VLP—artificial virus-like particlesCFA/IFA—complete Freund's adjuvant/incomplete Freund's adjuvant

An RCPL having 30 peptides, each 28 residues in length, that models theantigenic diversity of HIV-1 subtype B variants was synthesized in theform of MAP4. The antigenic properties of the RCPL was compared to aVCPL which also modeled the antigenic diversity of HIV-1 subtype Bvariants.

RCPL sequences (a period ‘.’ indicates that the residue unchanged fromthe first sequence listed): NNNTRKSIHIGPGQAFYATGDIIGDIRQ.....T..RL...RT..T..E......K S.....GV.......W....Q.......S.......R....R..FR......N... .......V......V..T..E.T....K.....RR.RM...RT............. G.......T....R.L.T..E...N........RG.P......L...........K .....T.V.L...RT..T..Q...............RM..........G.T.N... S.......P.....T....D.......K......G......R.WHT..Q....... S......V.L....V.........N........TG......R..FT..E......K G....R..R..L.R......E..............TR......L.T....T..... S......VT....RT..R.........K.....T...L....V.F...E....... ......G.R..........D....N..K......R..M...RT.....Q...N... S......VT....R.L.T..G............R...L....VW.T..E....... G.......R......L......T....K.....RGV.....RT..T......N... S....T.VP....R......Q......K........RL.W..T..T.........K ..K..R..........H...........S.....R..L...R...T..E...N... ..............T...R.Q.T..........TG.P....R.W.T..Q.......

A rabbit was immunized 3 times with the MAP4 VCPL and CFA (firstimmunization) or IFA (second immunization). The antibody titer of thesera was tested by ELISA using either the immunizing antigen, or theMAP4 RCPL as the immobilized peptide. The titer of rabbit serum in ELISAwith MAP4 VCPL on solid phase was 1:204,800, and with MAP4 RCPL on solidphase was 1:102,400.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. For example, chimericpeptide libraries can be designed to mimic the antigenic diversity ofother HIV proteins, or of proteins from other variable pathogens.Accordingly, other embodiments are within the scope of the followingclaims.

1-28. (canceled)
 29. A composition comprising a family of antigenicpeptides having amino acid sequences having antigenic similarity toamino acid sequences of a variable region of a pathogen protein, whereineach antigenic peptide in the family has at least one amino acidposition that varies relative to other antigenic peptides in the family.30. The composition of claim 29, wherein one amino acid residue occursmore frequently than another in the position that varies.
 31. Thecomposition of claim 29, wherein the family includes greater than 150mutually unique antigenic peptides.
 32. The composition of claim 29,wherein the family includes greater than 1,000 mutually unique antigenicpeptides.
 33. The composition of claim 29, wherein the family includesfewer than 100,000 mutually unique antigenic peptides.
 34. Thecomposition of claim 29, wherein the family includes fewer than 50,000mutually unique antigenic peptides.
 35. The composition of claim 29,wherein the family includes between 1,000 and 50,000 mutually uniqueantigenic peptides.
 36. The composition of claim 29, wherein thepathogen protein is HIV gp120.
 37. The composition of claim 36, whereinthe variable region is selected from the group consisting of the V1region, the V2 region, the V3 region, the V4 region and the V5 region.38. The composition of claim 37, wherein the family of antigenicpeptides includes sequences having antigenic similarity to sequencesfrom a subtype of HIV.
 39. The composition of claim 38, wherein thesubtype is selected from the group consisting of subtype A, subtype B,subtype C, subtype D, subtype F, subtype G, a recombinant subtype, asubtype of HIV group N, a subtype of HIV group O, and combinationsthereof.
 40. The composition of claim 29, wherein at least two membersof the family of antigenic peptides are mixed together.
 41. Thecomposition of claim 29, wherein the family of antigenic peptides areseparated according to sequence.
 42. The composition of claim 29,wherein the family includes a multiple antigenic peptide. 43-101.(canceled)
 102. A peptide library comprising a family of peptidesincluding the fragment:—N—N—T—R—K—X⁴—I—X⁵—X⁶—G—X⁷—G—X⁸—X⁹—X¹⁰—X¹¹—X¹²—T—X¹³—X¹⁴—I—X¹⁵—G—X¹⁶—I—R—wherein each X⁴-X¹⁶ is a fragment zero, one, two or three amino acidresidues in length.
 103. The peptide library of claim 102, wherein thefamily has antigenic similarity to the V3 region of HIV gp120.
 104. Thepeptide library of claim 103, wherein the family has antigenicsimilarity to the V3 region of HIV gp120 of HIV subtype B.
 105. Thepeptide library of claim 102, wherein the family of peptides have theformula:X¹—C—X²—R—P—X³—N—N—T—R—K—X⁴—I—X⁵—X⁶—G—X⁷—G—X⁸—X⁹—X¹⁰—X¹¹—X¹²—T—X¹³—X¹⁴—I—X¹⁵—G—X¹⁶—I—R—X¹⁷—A—X¹⁸—C—X¹⁹wherein each X¹-X¹⁹ is a fragment zero, one, two or three amino acidresidues in length.
 106. The peptide library of claim 105, wherein foreach peptide of the family, X¹ independently is N, T, or H.
 107. Thepeptide library of claim 105, wherein for each peptide of the family, X²independently is T or I.
 108. The peptide library of claim 105, whereinfor each peptide of the family, X³ independently is N, S, or G.
 109. Thepeptide library of claim 105, wherein for each peptide of the family, X⁴independently is S, G, or R.
 110. The peptide library of claim 105,wherein for each peptide of the family, X⁵ independently is H, P, N, Tor Y.
 111. The peptide library of claim 105, wherein for each peptide ofthe family, X⁶ independently is I or M.
 112. The peptide library ofclaim 105, wherein for each peptide of the family, X⁷ independently isP, L, or W.
 113. The peptide library of claim 105, wherein for eachpeptide of the family, X⁸ independently is R, Q, G or S.
 114. Thepeptide library of claim 105, wherein for each peptide of the family, X⁹independently is A, V or T.
 115. The peptide library of claim 105,wherein for each peptide of the family, X¹⁰ independently is F, W, or V.116. The peptide library of claim 105, wherein for each peptide of thefamily, X¹¹ independently is Y, F or H.
 117. The peptide library ofclaim 105, wherein for each peptide of the family, X¹² independently isT or A.
 118. The peptide library of claim 105, wherein for each peptideof the family, X¹³ independently is G, E or R.
 119. The peptide libraryof claim 105, wherein for each peptide of the family, X¹⁴ independentlyis E, Q, R or G.
 120. The peptide library of claim 105, wherein for eachpeptide of the family, X¹⁵ independently is I or T.
 121. The peptidelibrary of claim 105, wherein for each peptide of the family, X¹⁶independently is D or N.
 122. The peptide library of claim 105, whereinfor each peptide of the family, X¹⁷ independently is Q or K.
 123. Thepeptide library of claim 105, wherein for each peptide of the family,X¹⁸ independently is H or Y.
 124. The peptide library of claim 105,wherein for each peptide of the family, X¹⁹ independently is N or T.125. The peptide library of claim 105, wherein for each peptide of thefamily, X¹ independently is N, T or H; X² independently is T or I; X³independently is N, S, or G; X⁴ independently is S, G, or R; X⁵independently is H, P, N, T or Y; X⁶ independently is I or M; X⁷independently is P, L, or W; X⁸ independently is R, Q, G or S; X⁹independently is A, V or T; X¹⁰ independently is F, W, or V; X¹¹independently is Y, F or H; X¹² independently is T or A; X¹³independently is G, E or R; X¹⁴ independently is E, Q, R or G; X¹⁵independently is I or T; X¹⁶ independently is D or N; X¹⁷ independentlyis Q or K; X¹⁸ independently is H or Y; and X¹⁹ independently is N or T.126. The peptide library of claim 105, wherein at least two members ofthe family of peptides are mixed together.
 127. The peptide library ofclaim 105, wherein the family of peptides are separated according tosequence.
 128. The peptide library of claim 105, wherein the familyincludes greater than 150 mutually unique peptide sequences.
 129. Thepeptide library of claim 105, wherein the family includes fewer than100,000 mutually unique peptide sequences.
 130. The peptide library ofclaim 105, wherein the family includes fewer than 500 mutually uniquepeptide sequences, the sequences being representative of the entiresequence diversity available. 131-279. (canceled)